Cytotoxic and Antibiotic Cyclic Pentapeptide from an Endophytic

Article pubs.acs.org/JAFC

Cytotoxic and Antibiotic Cyclic Pentapeptide from an Endophytic Aspergillus tamarii of Ficus carica Yang-Min Ma,*,† Xi-Ai Liang,† Hong-Chi Zhang,†,§ and Rui Liu§ †

Key Laboratory of Auxiliary Chemistry & Technology for Chemical Industry, Ministry of Education. Shaanxi University of Science & Technology, Xi’an 710021, Shaanxi, China § College of Agronomy and Life Science, Shanxi Datong University, Datong 037009, Shanxi, China S Supporting Information *

ABSTRACT: A new cyclic pentapeptide, disulfide cyclo-(Leu-Val-Ile-Cys-Cys) (1), named malformin E, together with 13 known cyclic dipeptides, was isolated from the culture broth of endophytic fungus FR02 from the roots of Ficus carica. The strain FR02 was identified as Aspergillus tamarii on the basis of morphological characteristics and molecular analyses of internal transcribed spacer (ITS). Their structures were determined by the combination of 1D and 2D NMR spectroscopy, HRMS (ESI), UV, and Marfey’s analysis. Compound 1 exhibited strong cytotoxic activities against human cancer cell strains MCF-7 and A549 with IC50 values of 0.65 and 2.42 μM, respectively. It also displayed remarkable antimicrobial activities against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Penicillium chrysogenum, Candida albicans, and Fusarium solani with MIC values of 0.91, 0.45, 1.82, 0.91, 3.62, 7.24, and 7.24 μM, respectively. KEYWORDS: malformin E, Aspergillus tamarii, cytotoxicity, antimicrobial activity, cyclopeptide

■

chromatography (CC). Analytical thin-layer chromatography (TLC) was performed on silica gel GF254 plates, and spots were visualized by UV absorption and staining with Dragendorff’s reagent. HPLC analysis was run on a Waters C18 column (250 mm × 4.6 mm) on a Waters 2996 instrument (Waters Corp.). The bacteria (P. aeruginosa, E. coli, B. subtilis, and S. aureus) and the fungi (C. albicans, F. solani, and P. chrysogenum) were provided from the School of Food & Biological Engineering, Shaanxi University of Science & Technology (Xi’an, China). The cancer cell lines MCF-7 (breast cancer cells), HepG2 (liver cancer cells), and A549 (lung cancer cells) were provided from the College of Life Sciences, Northwest A&F University (Yangling, China). Fungal Material. The strain FR02 was isolated from the healthy roots of F. carica that was collected from Qinling Mountain in China’s Shaanxi province in September 2014. It was identified as A. tamarii on the basis of morphological characteristics and molecular analyses of ITS. The fungus was maintained on potato dextrose agar (PDA) slants and deposited at the College of Chemistry & Chemical Engineering, Shaanxi University of Science & Technology. Fermention, Extraction, and Isolation. Fermentation of endophytic fungus A. tamarii was carried out in Czapek’s medium composed of sucrose (30 g/L), MgSO4·7H2O (0.5 g/L), KH2PO4 (1.0 g/L), KCl (0.5 g/L), FeSO4 (0.01 g/L), and NaNO3 (3.0 g/L). Three pieces of the strain FR02 (diameter = 0.5 cm) from a mature PDA plate were inoculated into Erlenmeyer flasks (250 mL) containing 100 mL of the above sterile liquid medium, the fermentation flasks were cultured with an agitation rate of 135 rpm at 28 °C for 4 days to afford seed culture solution, and then 5 mL of seed culture solution was added to Erlenmeyer flasks (1000 mL) containing 300 mL of Czapek’s medium and cultured under the same condition for 15 days. The culture broth (80 L) was filtered and concentrated and then extracted with ethyl acetate three times to yield a crude extract (66.7 g). The

INTRODUCTION Endophytic fungi can produce structurally and biologically diverse natural products and have become an important source of novel structures and strong biological compounds.1,2 In recent years, more and more bioactive compounds from endophytic fungi of medical plants have been reported.3−5 Ficus carica is not only an edible plant but also a traditional medical plant; strain FR02 was isolated from the roots of F. carica, identified as Aspergillus tamarii. The metabolites displayed wide antibacterial and antifungal activities in vitro. In the course of investigating new strong bioactive compounds from the endophytic fungus A. tamarii, a new cyclic pentapeptide, disulfide cyclo-(Leu-Val-Ile-Cys-Cys) (1), named malformin E, together with 13 known cyclic dipeptides (2−14), fumitremorgin B (2), fumitremorgin C (3), cyclotryprostatin B (4), verruculogen (5), tryprostatin B (6), tryprostatin A (7), cyclotryprostatin A (8), cyclotryprostatin C (9), cyclo-(ProTrp) (10), cyclo-(N-methyl-Trp-Leu) (11), cyclotryprostatin D (12), 13-oxofumitremorgin B (13), and cyclo-(N-BenzylTrp-Pro) (14), were isolated. Compound 1 exhibited strong cytotoxicity to human cancer cell strains A549 and MCF-7. It also displayed remarkable antimicrobial activities against Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Fusarium solani, and Penicillium chrysogenum.

■

MATERIALS AND METHODS

General. 1D and 2D nuclear magnetic resonance (NMR) spectral data were obtained on a Bruker ADVANCE III 400 MHz spectrometer with CDCl3 and DMSO as solvents and tetramethylsilane (TMS) as internal standard. HRMS (ESI) was measured using a Bruker maXis 4G UHR-TOF. Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences Inc., Shanghai, China) were used for column © XXXX American Chemical Society

Received: March 4, 2016 Revised: April 18, 2016 Accepted: April 26, 2016

A

DOI: 10.1021/acs.jafc.6b01051 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Data of Compound 1 (DMSO-d6) δC

DEPT-135

173.5 50.8 41.3

C CH CH2

4 5 6 2-NH

24.9 23.2 22.2

CH CH3 CH3

7 8 9 10 11 8-NH

171.0 59.3 27.3 20.2 19.2

C CH CH CH3 CH3

12 13 14 15

173.2 58.5 34.4 25.2

C CH CH CH2

10.4 15.4

CH3 CH3

174.5 52.8 46.7

C CH CH2

position Leu

Val

Ile

1 2 3

16 17 13-NH Cys1

18 19 20

δH (J in Hz)

Cys2

21 22 23 22-NH

170.2 53.4 45.7

C CH CH2

H−1H COSY

HMBC

4.48 (td, J = 9.2, 6.2) 1.57−1.58 (m) 1.44−1.32 (m) 1.63−1.58 (m) 0.84 (s) 0.82 (s) 7.39 (d, J = 9.2)

3,2-NH 2

1, 3 1, 2, 4

3, 5, 6 4 4 2

3, 5, 6 4, 3 4 2, 7

3.94 (d, J = 9.8) 2.12−1.99 (m) 0.90 (d, J = 6.6) 0.87 (d, J = 6.6) 8.00 (d, J = 8.8)

9,8-NH 8 9 9 8

7, 8, 8, 8, 8,

3.89 (dd, J = 10.4, 3.6) 1.71 (dd, J = 15.1, 7.2) 1.46−1.37 (m) 1.23−1.08 (m) 0.82 (s) 0.79 (d, J = 6.8) 8.63 (d, J = 6.7)

14,17,13-NH 13, 15 14, 16

12, 14 13, 15, 17 14, 16, 13, 17

14 13 13

14, 15 13, 14, 15 13, 18

20,19-NH 19

18, 20 18, 19

19

19, 21

23,22-NH 22 22

21, 23 21, 22 22, 1

3.99 3.52 3.15 8.87

19-NH

1

(d, J = 3.5) (dd, J = 14.9, 3.2), (m) (d, J = 3.9)

4.72 (td, J = 10.9, 4.4) 3.28−3.10 (m) 7.12 (d, J = 11.0)

9 10, 11 9 9 12

Hz), 0.84 (s), 0.82 (s), 0.79 (d, J = 6.8 Hz); HRMS (ESI) m/z 552.2291 [M + Na]+ (calcd 552.2290). Biological Assays. Antimicrobial activity was evaluated in vitro by the serial 2-fold agar dilution method in the 96-well culture plates.6 The bacteria and pathogenic fungi used in this study were S. aureus, B. subtilis, E. coli, P. aeruginosa, C. albicans, F. solani, and P. chrysogenum, respectively. Briefly, 100 μL of compound 1 dissolved in DMSO (concentrations of 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, and 0.98 μg/mL) was added to each well, respectively, and then 100 μL of bacteria or fungus suspension at a density of 106 CFU/mL was inoculated to each well. All test plates were then incubated at 37 °C for 24 h for bacteria and at 28 °C for 48 h for fungi. The tested strains were incubated in liquid media, Lysogeny broth (LB) medium for bacteria and Sabouraud dextrose broth (SDB) for fungi. The result of assay was recorded by the values of the minimal inhibition concentration (MIC). Gentamicin was used as a positive control against bacteria and nystatin as a positive control for the antifungal assay. The cytotoxic effect of compounds in vitro was preliminarily assayed on MCF-7, HepG2, and A549 cell lines by the MTT (3-(4,5dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide) method.7 Three cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. The cells were subcultured twice a week in medium, RPMI-1640 medium for MCF-7 cells and Dulbecco’s modified Eagle medium (DMEM) for HepG2 and A549 cells. Normally, 200 μL of these cell suspensions (5 × 104 cells mL−1) was plated in 96-well culture plates at 37 °C. After 12 h, then 20 μL of

crude extract was subjected to chromatography over a silica gel column followed by a stepwise gradient elution of petroleum ether in ethyl acetate or ethyl acetate in methanol to afford seven fractions (fractions 1−7). Fraction 2 was further separated by chromatography over a silica gel column eluting with ethyl acetate/methanol (100:1 to 50:1, v/v), and a Sephadex LH-20 column eluting with ethyl acetate/methanol (1:1) afforded compound 2 (37 mg), compound 3 (29 mg), compound 4 (19 mg), and compound 5 (28 mg). Fraction 3 was further separated by chromatography over a silica gel column eluting with ethyl acetate/methanol (50:1 to 25:2, v/v) to afford compound 6 (32 mg), compound 1 (12 mg), compound 7 (11 mg), compound 8 (9 mg), compound 9 (9 mg), and compound 10 (48 mg). Fraction 4 was further separated by chromatography over a silica gel column eluting with ethyl acetate/methanol (50:1 to 25:3, v/v) to afford compound 11 (17 mg), compound 12 (7 mg), compound 13 (15 mg), and compound 14 (23 mg). Compound 1: white amorphous powder; 13C NMR (DMSO-d6) (Table 1) δ 174.5, 173.5, 173.2, 170.2, 171.0, 59.3, 58.5, 53.4, 52.8, 50.8, 46.7, 45.7, 41.3, 34.4, 27.3, 25.2, 24.9, 23.2, 22.2, 20.2, 19.2, 15.4, 10.4; 1H NMR (DMSO-d6) δ 8.87 (d, J = 3.9 Hz), 8.63 (d, J = 6.7 Hz), 8.00 (d, J = 8.8 Hz), 7.39 (d, J = 9.2 Hz), 7.12 (d, J = 11.0 Hz), 4.72 (td, J = 10.9, 4.4 Hz), 4.48 (td, J = 9.2, 6.2 Hz), 3.99 (d, J = 3.5 Hz), 3.94 (d, J = 9.8 Hz), 3.89 (dd, J = 10.4, 3.6 Hz), 3.52 (dd, J = 14.9, 3.2 Hz), 3.28−3.10 (m), 3.15 (m), 2.12−1.99 (m), 1.63−1.58 (m), 1.71 (dd, J = 15.1, 7.2 Hz), 1.57−1.58 (m), 1.46−1.37 (m), 1.44−1.32 (m), 1.23−1.08 (m), 0.90 (d, J = 6.6 Hz), 0.87 (d, J = 6.6 B

DOI: 10.1021/acs.jafc.6b01051 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of compounds 1−14.

NMR spectra revealed five carbonyl signals (δC 174.5, 173.5, 173.2, 171.0, 170.2) and five amide protons signals (δH 8.87, 8.63, 8.00, 7.39, 7.12). According to the preliminary estimation, compound 1 may be the peptide class of compounds. The 1 H−1H COSY (Figure 2) experiment showed the five amino

compound 1 dissolved in DMSO (concentrations of 10 mM, 1 mM, 100 μM, 10 μM, and 1 μM) was added to each well respectively and cultured at 37 °C for 24 h. Then the MTT was dissolved in saline to 5 mg/mL, and 20 μL of this solution was added to each well. After 4 h, 100 μL of DMSO was added to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. Positive controls were either adriamycin or 5-fluorouracil. Reed and Muench’s method was used to calculated IC50 values.8

■

RESULTS AND DISCUSSION Structure Elucidation. Marfey’s analysis method was performed to measured the stereochemistry of the amino acid residues.9 Briefly, 2 mg of compound 1 dissolved in 2 mL of 6 mol/L HCl was heated in a sealed glass tube at 110 °C; after 24 h, the pH of the mixture was adjusted to 6.0 with NaOH at room temperature, then concentrated and redissolved in 2 mL of distilled water, and finally reacted with 1-fluoro-2,4dinitrophenyl-5-L-alaninamide (FDAA). The result was analyzed by reversed-phase HPLC and compared with standard amino acids. The analysis revealed all of the amino acid residues were L-configurations. The molecular formula of compound 1 (Figure 1) was determined as C23H39N5O5S2 according to the HRMS (ESI) at m/z 552.2291 [M + Na]+ (calcd 552.2290). The 1H and 13C

Figure 2. Key 1H−1H COSY and HMBC correlations of compound 1.

acid residues as leucine, valine, isoleucine, and two cysteines. 1 H−1H COSY correlations of H-2/2-NH and H-3, H-5, H-6/ H-4, as well as HSQC correlation and δC 173.5, 50.8, 41.3, 24.9, 23.2, and 22.2 revealed the leucine residue. H-8/8-NH and H-9, H-10, H-11/H-9, as well as HSQC correlation and δC 171.0, 59.3, 27.3, 20.2, and 19.2, revealed the valine residue. H-13/13NH and H-14, H-15, H-17/H-14, H-15/H-16, as well as C

DOI: 10.1021/acs.jafc.6b01051 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry Table 2. Antimicrobial Activity of Compound 1 MIC (μM) Gram-positive bacteria

a

Gram-negative bacteria

fungi

compound

B. subtilis

S. aureus

E. coli

P. aeruginosa

C. albicans

F. solani

P. chrysogenum

1 gentamicin nystatin

0.91 4.06 −

0.45 2.03 −

0.91 4.06 −

1.82 2.03 −

7.24 −a 6.16

7.24 − 6.72

3.62 − 13.5

“−”, not set test.

■

HSQC correlation and δC 173.2, 58.5, 34.4, 25.2, 10.4, and 15.4, revealed the isoleucine residue. H-19/19-NH and H-20, H-22/ 22-NH and H-23, as well as HSQC correlation and δC 174.5, 52.8, 46.7, 170.2, 53.4, and 45.7, revealed two cysteine residues. The amino acid sequence was conformed mainly by HMBC correlations of the carbonyl carbon of one amino acid residue with the amide protons of the neighboring residue. As shown in Figure 2, we could see the HMBC correlations of δH 7.39/δC 171.0, δH 8.00/δC 173.2, δH 8.63/δC 174.5, δH 8.87/δC 170.2, and δH 7.12/δC 173.5. Thus, from the above-mentioned evidence compound 1 was confirmed to be disulfide cyclo(Leu-Val-Ile-Cys-Cys). By detailed physicochemical properties and spectroscopic analyses, as well as by comparisons of the literature data, the structures of compounds 2−14 (Figure 1) were identified as fumitremorgin B (2),10,11 fumitremorgin C (3),12 cyclotryprostatin B (4),13 verruculogen (5),14 tryprostatin B (6),12 tryprostatin A (7),12 cyclotryprostatin A (8),13 cyclotryprostatin C (9),13 cyclo-(Pro-Trp) (10),15 cyclo-(N-methyl-Trp-Leu) (11),16 cyclotryprostatin D (12),13 13-oxofumitremorgin B (13),17 and cyclo-(N-Benzyl-Trp-Pro) (14),18 respectively. Biological Activities. The result of antimicrobial activity showed compound 1 has potent antimicrobial activity. It displayed significant growth inhibition against B. subtilis, S. aureus, P. aeruginosa, and E. coli with MIC values of 0.91, 0.45, 1.82, and 0.91 μM, respectively. Meanwhile, it also has strong antifungal activities against P. chrysogenum, C. albicans, and F. solani with MIC values of 3.62, 7.24, and 7.24 μM, respectively (Table 2). In the MTT bioassay, compound 1 exhibited strong cytotoxicity to human cancer cell strains A549 and MCF-7 with IC50 values of 2.42 and 0.65 μM, respectively. It also displayed slight activity with an IC50 value of 36.02 μM on HepG2 cells (Table 3).

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01051. Various spectra of compound 1; strain identification of FR02; NMR of compounds 2−14 (PDF)

■

*(Y.-M.M.) E-mail: [email protected]. Phone: +86-2986168312. Fax: +86-29-86168312. Funding

This research was financially supported by the Natural Science Basic Research Plan in Shaanxi Province of China (2014JZ003). Notes

The authors declare no competing financial interest.

■

MCF-7

A549

HepG2

0.65 1.24 3.35

2.42 4.28 1.62

36.02 16.78 4.31

REFERENCES

(1) Li, H.; Xiao, J.; Gao, Y. Q.; Tang, J. J.; Zhang, A. L.; Gao, J. M. Chaetoglobosins from Chaetomium globosum, an endophytic fungus in Ginkgo biloba, and their phytotoxic and cytotoxic activities. J. Agric. Food Chem. 2014, 62 (17), 3734−3741. (2) Strobel, G.; Daisy, B. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 2003, 67, 491− 502. (3) Liu, D.; Li, X. M.; Meng, L.; Li, C. S.; Gao, S. S.; Shang, Z.; Proksch, P.; Huang, C. G.; Wang, B. G. Nigerapyrones A−H, a-pyrone derivatives from the marine mangrove-derived endophytic fungus Aspergillus niger MA-132. J. Nat. Prod. 2011, 74, 1787−1791. (4) Luo, J.; Liu, X. Y.; Li, E.; Guo, L. D.; Che, Y. S. Arundinols A−C and arundinones A and B from the plant endophytic fungus Microsphaeropsis arundinis. J. Nat. Prod. 2013, 76, 107−112. (5) Ortega, H. E.; Graupner, P. R.; Asai, Y.; TenDyke, K.; Qiu, D.; Shen, Y. Y.; Rios, N.; Arnold, A. E.; Coley, P. D.; Kursar, T. A.; Gerwick, W. H.; Cubilla-Rios, L. Mycoleptodiscins A and B, cytotoxic alkaloids from the endophytic fungus Mycoleptodiscus sp. F0194. J. Nat. Prod. 2013, 76, 741−744. (6) Appendino, G.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G.; Stavri, M.; Smith, E.; Rahman, M. Antibacterial cannabinoids from Cannabis sativa: a structure activity study. J. Nat. Prod. 2008, 71, 1427−1430. (7) Zhang, J. T. Experiment Methods of Mordern Pharmacology; Beijing Medical University and China Xie-he Medical University Joint Publishing House, 1998. (8) Reed, L. J.; Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 1938, 27, 493−497. (9) Fujii, K.; Shimoya, T.; Ikai, Y.; Oka, H.; Harada, K.-i. Further application of advanced Marfey’s method for determination of absolute configuration of primary amino compound. Tetrahedron Lett. 1998, 39, 2579−2582. (10) Yamazaki, M.; Sasago, K.; Miyaki, K. The structure of fumitremorgin B (FTB), a tremorgenic toxin from Aspergillus f umigatus Fres. J. Chem. Soc., Chem. Commun. 1974, 10, 408−409.

IC50 (μM) compound

AUTHOR INFORMATION

Corresponding Author

Table 3. In Vitro Cytotoxicity Evaluation of Compound 1 against Three Tumor Cell Lines

1 adriamycin 5-fluorouracil

ASSOCIATED CONTENT

In conclusion, a new cyclic pentapetide, malformin E, together with 13 known cyclic peptides, was obtained from the culture broth of endophytic fungus A. tamarii from the roots of F. carica. The results revealed that endophytic fungus A. tamarii is a rich source of potent bioactive cyclic peptides. Malformin E showed significant antimicrobial and cytotoxic activities in vitro, which indicated malformin E could be a promising candidate for new antitumor and antimicrobial agents in the agricultural and medical industries. D

DOI: 10.1021/acs.jafc.6b01051 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry (11) Masako, N.; Shin-ichi, K.; Mitsuya, H.; Tomohiko, K.; Tohru, H. Total synthesis of fumitremorgin B. Tetrahedron Lett. 1986, 27, 6217−6220. (12) Cui, C. B.; Kakeya, H.; Osada, H. Novel mammalian cell cycle inhibitor, tryprostatins A, B and other diketopiperazines produced by Aspergillus f umigatus II. Physicochemical properties and structures. J. Antibiot. 1996, 49, 534−540. (13) Cui, C. B.; Gakeya, H.; Osada, H. Novel mammalian cell cycle inhibitor, cyclotryprostatins A−D, produced by Aspergillus f ionigatus, which inhibit mammalian cell cycle at G2/M phase. Tetrahedron 1997, 53, 59−72. (14) Fayos, J.; Lokensgard, D.; Clardy, J.; Cole, R. J.; Kirksey, J. W. Structure of verruculogen, a tremor producing peroxide from Penicillium verruculosum. J. Am. Chem. Soc. 1974, 96, 6785−6787. (15) Steyn, P. S. The structures of five diketopiperazines from Aspergillus ustus. Tetrahedron 1973, 29, 107−120. (16) Tone, H.; Sato, S.; Sato, H.; Tamura, K.; Tamada, S. Preparation of 3-(3-indolylmethyl) piperazine derivatives as superoxide radical inhibitors for prevention and treatment of nephritis. PCT Int. Appl. 1990, 509−515. (17) Fujimoto, H.; Fujimaki, T.; Okuyama, E.; Yamazaki, M. Immunosuppressive constituents from an Ascomycetes sordaria gondaensis. Mycotoxins 2000, 50 (2), 93−99. (18) Wang, H.; Usui, T.; Osada, H.; Ganesan, A. Synthesis and evaluation of tryprostatin B and demethoxy fumitremorgin C analogues. J. Med. Chem. 2000, 43, 1577−1585.

E

DOI: 10.1021/acs.jafc.6b01051 J. Agric. Food Chem. XXXX, XXX, XXX−XXX